Ethanol is widely used in potable alcoholic beverages, as a solvent for extraction or chemical synthesis, or as a fuel additive or straight fuel. Alcohol is mainly produced through fermentation by yeast of plant-derived material that is rich in carbohydrates. Starch-rich grains of cereals, including but not limited to wheat, corn, barley, rye, rice, oats, triticale, spelt, sorghum, or pearl millet, can be used as a starting material for ethanol production. The processing of cereals for ethanol production generates co-products, such as for instance dried distillers grain and solubles (DDGS), which are used as low-value animal feed or burned to recover heat energy. There is a need for diversification of the co-products of ethanol production, especially co-products with a high added value.
Cereals contain 5-10% of arabinoxylan, which together with starch, cellulose and β-glucan constitute the most abundant cereal carbohydrates. Arabinoxylan comprises a main chain of β-1,4-linked D-xylopyranosyl units to which O-2 and/or O-3 α-L-arabino-furanosyl units are linked (Gruppen et al. 1992). In a typical arabinoxylan, unsubstituted, monosubstituted and disubstituted xylose residues occur (see FIG. 1). In addition, the arabinose side chains can be covalently linked to acetyl, methylglucuronic acid, or feruloyl substituents, the latter being capable to form ferulic acid dimer cross-links. Arabinoxylans in cereals are either water-extractable or water-unextractable (Gruppen et al. 1992; Courtin and Delcour, 2001). Water-unextractable arabinoxylans (WU-AX) can be partially solubilised under alkaline conditions or by using enzymes, such as endoxylanases. WU-AX bind large amounts of water. The water-extractable arabinoxylans (WE-AX) have very high molecular masses (up to 800,000 Dalton) depending on the source and extraction method, and have an extraordinary viscosity forming potential. Arabinoxylans can not be fermented by standard strains of the yeast Saccharomyces cerevisiae, and therefore do not contribute to the production of ethanol by fermentation of cereals.
Arabinoxylo-oligosaccharides (AXOS), oligosaccharides derived from arabinoxylan, have been shown to exert prebiotic properties and could therefore be an interesting co-product of ethanol production. Prebiotics are compounds, usually non-glucosidic oligosaccharides, that can not be digested by enzymes of the upper gastro-intestinal tract but are fermented selectively by some types of intestinal bacteria in the large intestine (Gibson and Roberfroid, 1995; Roberfroid, 1988; Van Loo, 2004). The presence of prebiotics in the diet causes a shift in the composition of the intestinal bacterial population, typically characterised by a relative increase in Lactobacillus and Bifidobacterium species. This shift in the microbiota of the intestine is associated with improved overall health, reduced gut infections, increased levels of intestinal short chain fatty acids, better absorption of minerals, and suppression of colon cancer initiation (Van Loo, 2004). A preparation consisting predominantly of AXOS with a degree of polymerisation (DP) of 3-5 (arabinosylxylobiose, arabinosylxylotriose, arabinosylxylotetraose, and diarabinosylxylotetraose) has been shown to exert prebiotic effects and to increase the levels of Bifidobacteria in the intestines of rats and mice (Yamada et al., 1993). It has further been demonstrated that AXOS preparations with an average DP ranging between 5 and 50 have particularly strong prebiotic properties, as evidenced by tests performed on chickens, rats and humans (WO06/002495). AXOS have also been shown to improve the growth performance and food utilisation of monogastric animals (WO03/015533). AXOS-containing preparations can therefore find wide uses as ingredients for food, beverage and feed products.
Up to now no economically viable method for the production of AXOS has been developed. The method developed by Yamada et al. (1993) involves chemical extraction of arabinoxylan using a concentrated alkaline solution, followed by neutralisation, removal of the salts, enzymic hydrolysis with endoxylanase, and chromatography on a carbon column (Yamada et al., 1993). The main drawback of this method is that the alkaline extraction of arabinoxylan is environment-unfriendly, and requires costly removal of the minerals by extensive dialysis or ultrafiltration before enzymic hydrolysis can be performed. Another method to produce AXOS involves hydrothermal autohydrolysis of hardwood or brewery spent grain. In this method a suspension of plant material is heated in a special reactor at 150-190° C. for 20-60 min (EP 0265970B1; Kabel et al., 2002; Carvalheiro et al., 2004). The drawback of this method is that, due to the high reaction temperature, side products are produced that are undesirable for food purposes, such as furfural, hydroxymethylfurfural and levulinic acid (Carvalheiro et al., 2004). Other methods have used endoxylanase enzymes to solubilise AXOS from WU-AX, in particular from cereal bran which is known to be rich in WU-AX (Delcour et al. 1999). Several related procedures have been described in which bran is first treated with an alpha amylase to convert starch in soluble maltodextrins, whereafter AXOS is released from the insoluble fraction through the action of endoxylanase (Maes et al 2004; Swennen et al. 2006; WO2006/027529). Patent US2002/0037331 teaches a method to extract AXOS from bran consisting of extrusion followed by treatment with endoxylanase and amylase, further followed by ultrafiltration. The drawback of bran-based methods are the relatively low recovery of AXOS and the low value of the abundantly generated co-products. The present invention provides a method allowing the co-production of AXOS in an ethanol production process using arabinoxylan-containing cereal material as feedstock. The method according to the present invention comprises the exposure of the cereal material to an appropriate endoxylanase enzymatic activity in order to solubilise a substantial portion of the water-unextractable arabinoxylans comprised therein. The co-production of ethanol and AXOS has the advantage that it allows a more efficient use of the infrastructure, energy input and raw materials, while providing an AXOS preparation suitable for use as a food or animal feed additive. Moreover, the co-production of AXOS according to the present invention has no negative impact on the yield of the ethanol production.
The most commonly used method for production of ethanol from cereals is the so-called dry milling process. In this process whole grains are used as an input, in contrast to the wet milling process which uses purified starch or starch-rich endosperm. The steps in conventional dry milling include (FIG. 2):                Milling: grinding of the whole grains to obtain a fine powder.        Mashing: mixing of the milled grain with water to obtain a mash or slurry.        Liquefaction: conversion of the starch to maltodextrins by addition of an alpha-amylase enzyme to the mash, by which the mash is turned into a so-called “liquefied mash”.        Saccharification: conversion of the maltodextrins in the liquefied mash to glucose and maltose by addition of an amyloglucosidase or glucoamylase enzyme, by which the liquefied mash is turned into a saccharified mash. Typically, an enzyme preparation comprising endoxylanase activity is added prior to or during the saccharification. Alternatively, said enzyme preparation is added at the start of the fermentation step.        Fermentation: conversion of the glucose and maltose in the saccharified mash to ethanol and carbon dioxide through the action of a microorganism, e.g. the yeast Saccharomyces cerevisiae, by which the saccharified mash is turned into a fermented mash. Most often fermentation is done concurrently with saccharification in a process called simultaneous saccharification and fermentation or SSF. In some processes, liquefaction, saccharification and fermentation are performed concurrently using special enzyme blends such as STARGEN™ from the company Genencor.        Distillation: separation of ethanol from the aqueous phase. The remaining slurry consisting of aqueous phase and solids is called “whole stillage”. The resulting ethanol fraction can be further dehydrated to remove residual water, or otherwise further refined.        Centrifugation: separation of the whole stillage into a solid fraction, called “distillers wet grains”, and a soluble fraction, called “thin stillage”.        Evaporation: concentration of the thin stillage to obtain a so-called “condensed thin stillage” or “condensed distillers solubles”.        Drying: drying of the wet grains to obtain “distillers dried grains (DDG)”, or drying of a mixture of distillers wet grains and condensed distillers solubles to obtain “distillers dried grains and solubles (DDGS)”.        
Further information on ethanol production processes be can be found in e.g. Lyons et al (1995) and Ward and Singh (2002). Recently, it is considered to also use bran or bran rich material, such as shorts, as a starting material instead or in combination with whole grains in the bioethanol production. Any of such starting materials are suitable for the purpose of the present invention.
Xylanases are sometimes used in ethanol production, and such enzymes are either added to the mash after liquefaction, during fermentation, or to the whole stillage or thin stillage. The purpose of the addition of xylanases is to reduce the viscosity of the slurries and liquids, and hence to allow increasing the gravity of slurries, facilitating pumping and to increase the efficiency of heat transfer (WO02/38786; WO2004/087889, Sorensen et al. 2006). Since viscosity in cereals is conferred by WE-AX and not by WU-AX (Meuser et al. 1986), the xylanases used in a conventional cereal based ethanol production process are either endoxylanases with a high selectivity for WE-AX, or exoxylanases, such as beta-xylosidases. This practice does not allow the solubilisation of a significant amount of water-unextractable arabinoxylans and hence does not result in the isolation of soluble arabinoxylans with an acceptable yield.